Potato tuber flesh colour is a quality trait dependent on the types and levels of carotenoids. These, together with many plant growth regulators, sterols and terpenes, are isoprenoid derived compounds. Even though carotenoids have been extensively studied due to their importance in photosynthesis and nutrition, a clear understanding concerning the regulation of their accumulation and localisation in plant storage organs is lacking. The present study describes two strategies to investigate the regulation of carotenoid biosynthesis in potato. Firstly, in order to localise the site of synthesis of tuber carotenoids, stable transgenic potato plants were generated. These expressed two key carotenoid biosynthetic genes, PHYTOENE SYNTHASE 2 (PSY2) and β-CAROTENE HYDROXYLASE 2 (CrtRb2), fused to red fluorescent protein (RFP). The expression and carotenoid levels were significantly increased, confirming functionality of the protein-RFP ensembles. Confocal microscopy on developing tuber tissue made it possible to obtain images of PSY2-RFP and CrtRb2-RFP localisation into amyloplasts. Secondly, the effect of carotenoid degradation was addressed as a potential regulator of accumulation, based on the recent discovery that a CAROTENOID CLEAVAGE DIOXYGENASE 4 (CCD4) reduces carotenoid levels in potato tubers. CCD8 is another important member of the CCD gene family, with a role in strigolactone biosynthesis. In this study, CCD8-RNAi knock out lines were characterised. Although CCD8 down-regulation did not significantly affect carotenoid content, a range of interesting above and below ground phenotypes was observed. Importantly, the study showed that the CCD8 gene product impacts on stolon branching, tuber development and tuber dormancy.